Interactions of the super complexes: When mTORC1 meets the proteasome.

Homeostatic regulation of energy and metabolic status requires that anabolic and catabolic signaling pathways be precisely regulated and coordinated. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a mega protein complex that promotes energy-consuming anabolic processes of protein and nucleic acid synthesis as well lipogenesis in times of energy and nutrient abundance. However, it is best characterized as the regulator of steps leading to protein synthesis. The ubiquitin-proteasome proteolytic system (UPS) is a major intracellular proteolytic system whose activity is increased during periods of nutrient scarcity and in muscle wasting conditions such as cachexia. Recent studies have examined the impact of mTORC1 on levels and functions of the 26S proteasome, the mega protease complex of the UPS. Here we first briefly review current understanding of the regulation of mTORC1, the UPS, and the 26S proteasome complex. We then review evidence of the effect of each complex on the abundance and functions of the other. Given the fact that drugs that inhibit either complex are either in clinical trials or are approved for treatment of cancer, a muscle wasting condition, we identify studying the effect of combinatory mTORC1-proteasome inhibition on skeletal muscle mass and health as a critical area requiring investigation.

Physical function-derived cut-points for the diagnosis of sarcopenia and dynapenia from the Canadian longitudinal study on aging.

BACKGROUND: Aging is associated with sarcopenia (low muscle mass) and dynapenia (low muscle strength) leading to disability and mortality. Widely used previous cut-points for sarcopenia were established from dated, small, or pooled cohorts. We aimed to identify cut-points of low strength as a determinant of impaired physical performance and cut-points of low appendicular lean mass (ALM) as a predictor of low strength in a single, large, and contemporary cohort of community-dwelling older adults and compare these criteria with others. METHODS: Cross-sectional analyses were conducted on baseline data from 4725 and 4363 community-dwelling men and women (65-86 years, 96.8% Caucasian) of the Canadian longitudinal study on aging comprehensive cohort. Physical performance was evaluated from gait speed, timed up-and-go, chair rise, and balance tests; a weighted-sum score was computed using factor analysis. Strength was measured by handgrip dynamometry; ALM, by dual-energy X-ray absorptiometry and ALM index (ALMI; kg/m2 ), was calculated. Classification and regression tree analyses determined optimal sex-specific cut-points of ALMI predicting low strength and of strength predicting impaired physical performance (score < 1.5 SD below the sex-specific mean). RESULTS: Modest associations were found between ALMI and strength and between strength and physical performance score in both sexes. ALMI was not an independent predictor of physical performance score. Cut-points of <33.1 and <20.4 kg were found to define dynapenia in men and in women, respectively, corresponding to 21.5% and 24.0% prevalence rates. Sarcopenia cut-points were <7.76 kg/m2 in men and <5.72 kg/m2 in women; prevalence rates of 21.7% and 13.7%. Overall, 8.3% of men and 5.5% of women had sarco-dynapenia. Sarcopenic were older and had lower fat mass and body mass index (BMI) than non-sarcopenic participants. While the agreement between current criteria and the updated European Working Group for Sarcopenia in Older Persons recommendations was fair, we found only slight agreement with the Foundation for the National Institute of Health sarcopenia project. Older persons identified with sarcopenia as per the Foundation for the National Institute of Health criteria (using ALM/BMI as the index) have higher BMI and fat mass compared with non-sarcopenic and have normal ALMI as per our criteria. CONCLUSIONS: The proposed function-derived cut-points established from this single, large, and contemporary Canadian cohort should be used for the identification of sarcopenia and dynapenia in Caucasian older adults. We advise on using criteria based on ALMI in the diagnosis of sarcopenia. The modest agreement between sarcopenia and dynapenia denotes potential distinct health implications justifying to study both components separately.

The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation-associated cachectic muscle wasting.

Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.

Ubiquitin Ligase Huwe1 Modulates Spermatogenesis by Regulating Spermatogonial Differentiation and Entry into Meiosis.

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Role of the deubiquitinating enzyme ubiquitin-specific protease-14 in proteostasis in renal cells.

Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus inhibition of Usp14 may enhance degradation of misfolded proteins and attenuate cell injury. In GEC, the reporter proteins GFPu (a "misfolded" protein) and CD3δ (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3δ-yellow fluorescent protein (YFP), but accelerated degradation of GFPu, and the Usp14-directed inhibitor IU1 further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3δ-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor tunicamycin and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury.

Deubiquitinating enzymes in skeletal muscle atrophy-An essential role for USP19.

The ubiquitin proteasome system is well recognized to be involved in mediating muscle atrophy in response to diverse catabolic conditions. To date, almost all of the genes that have been implicated are ubiquitin ligases. Although ubiquitination is modulated also by deubiquitinating enzymes, the roles of these enzymes in muscle wasting remains largely unexplored. In this article, the potential roles of deubiquitinating enzymes in regulating muscle size are discussed. This is followed by a review of the roles described for USP19, the deubiquitinating enzyme that has been most studied in muscle wasting. This enzyme is upregulated in muscle in many catabolic conditions and its inactivation leads to protection from muscle loss induced by stimuli that are common in many illnesses causing cachexia. It can regulate both protein synthesis and protein degradation as well as myogenesis, thereby modulating the key processes that control muscle mass. Roles for other deubiquitinating enzymes remain possible and to be explored.

Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling.

The USP19 deubiquitinating enzyme modulates the expression of myogenin and myofibrillar proteins in L6 muscle cells. This raised the possibility that USP19 might regulate muscle cell differentiation. We therefore tested the effects of adenoviral-mediated overexpression or small interfering RNA (siRNA)-mediated silencing of either the cytoplasmic or endoplasmic reticulum (ER)-localized isoforms of USP19. Only the ER-localized isoform of USP19 (USP19-ER) modulated myoblast fusion as well as the expression of myogenin and myofibrillar proteins, and these effects were also dependent on USP19 catalytic activity. USP19-ER inhibited muscle cell differentiation and the induction of CHOP, a transcription factor in the unfolded-protein response (UPR) that is activated during differentiation. Inducing the UPR by creating mild ER stress with thapsigargin was able to reverse the defect in myoblast fusion caused by the overexpression of USP19-ER, suggesting strongly that USP19 exerts its effects on fusion through its effects on UPR signaling. USP19 also functions similarly in vivo, as USP19(-/-) mice display improved muscle regeneration concomitant with enhanced expression of CHOP. Collectively these results implicate a deubiquitinating enzyme as a regulator of the UPR. They also suggest that inhibition of USP19 may be a therapeutic approach for the enhancement of muscle growth following injury.

A central role for ubiquitination within a circadian clock protein modification code.

Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.

Deubiquitinases in skeletal muscle atrophy.

The ubiquitin proteasome system plays a critical role in skeletal muscle atrophy. A large body of research has revealed that many ubiquitin ligases are induced and play an important role in mediating the wasting. However, relatively little is known about the roles of deubiquitinases in this process. Although it might be expected that deubiquitinases would be downregulated in atrophying muscles to promote ubiquitination and degradation of muscle proteins, this has not to date been demonstrated. Instead several deubiquitinases are induced in atrophying muscle, in particular USP19 and USP14. USP19, USP2 and A20 are also implicated in myogenesis. USP19 has been most studied to date. Its expression is increased in both systemic and disuse forms of atrophy and can be regulated through a p38 MAP kinase signaling pathway. In cultured muscle cells, it decreases the expression of myofibrillar proteins by apparently suppressing their transcription indicating that the ubiquitin proteasome system may be activated in skeletal muscle to not only increase protein degradation, but also to suppress protein synthesis. Deubiquitinases may be upregulated in atrophy in order to maintain the pool of free ubiquitin required for the increased overall conjugation and degradation of muscle proteins as well as to regulate the stability and function of proteins that are essential in mediating the wasting. Although deubiquitinases are not well studied, these early insights indicate that some of these enzymes play important roles and may be therapeutic targets for the prevention and treatment of muscle atrophy. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

Complement modulates the function of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells.

In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.

Ataxin-3 deubiquitination is coupled to Parkin ubiquitination via E2 ubiquitin-conjugating enzyme.

We reported previously that parkin, a Parkinson disease-associated E3 ubiquitin-ligase interacts with ataxin-3, a deubiquitinating enzyme associated with Machado-Joseph disease. Ataxin-3 was found to counteract parkin self-ubiquitination both in vitro and in cells. Moreover, ataxin-3-dependent deubiquitination of parkin required the catalytic cysteine 14 in ataxin-3, although the precise mechanism remained unclear. We report here that ataxin-3 interferes with the attachment of ubiquitin (Ub) onto parkin in real-time during conjugation but is unable to hydrolyze previously assembled parkin-Ub conjugates. The mechanism involves an ataxin-3-dependent stabilization of the complex between parkin and the E2 Ub-conjugating enzyme, which impedes the efficient charging of the E2 with Ub. Moreover, within this complex, the transfer of Ub from the E2 is diverted away from parkin and onto ataxin-3, further explaining how ataxin-3 deubiquitination is coupled to parkin ubiquitination. Taken together, our findings reveal an unexpected convergence upon the E2 Ub-conjugating enzyme in the regulation of an E3/deubiquitinating enzyme pair, with important implications for the function of parkin and ataxin-3, two proteins responsible for closely related neurodegenerative diseases.

Targeting protein synthesis in a Myc/mTOR-driven model of anorexia-cachexia syndrome delays its onset and prolongs survival.

Anorexia-cachexia syndrome (ACS) is a major determinant of cancer-related death that causes progressive body weight loss due to depletion of skeletal muscle mass and body fat. Here, we report the development of a novel preclinical murine model of ACS in which lymphomas harbor elevated Myc and activated mTOR signaling. The ACS phenotype in this model correlated with deregulated expression of a number of cytokines, including elevated levels of interleukin-10 which was under the direct translational control of mTOR. Notably, pharmacologic intervention to impair protein synthesis restored cytokine production to near-normal levels, delayed ACS progression, and extended host survival. Together, our findings suggest a new paradigm to treat ACS by strategies which target protein synthesis to block the production of procachexic factors.

Identification of distinctive patterns of USP19-mediated growth regulation in normal and malignant cells.

We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27(Kip1). To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27(Kip1). However, in contrast to previous findings in fibroblasts, the stabilization of p27(Kip1) upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27(Kip1) and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27(Kip1) levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.

Mechanisms involved in 3',5'-cyclic adenosine monophosphate-mediated inhibition of the ubiquitin-proteasome system in skeletal muscle.

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men.

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.

The UPS in diabetes and obesity.

Type 2 diabetes is caused by defects in both insulin signaling and insulin secretion. Though the role of the ubiquitin proteasome system (UPS) in the pathogenesis of type 2 diabetes remains largely unexplored, the few examples present in the literature are interesting and suggest targets for drug development. Studies indicate that insulin resistance can be induced by stimulating the degradation of important molecules in the insulin signaling pathway, in particular the insulin receptor substrate proteins IRS1, IRS2 and the kinase AKT1 (Akt). In addition, a defect in insulin secretion could occur due to UPS-mediated degradation of IRS2 in the beta-cells of the pancreas. The UPS also appears to be involved in regulating lipid synthesis in adipocytes and lipid production by the liver and could influence the development of obesity. Other possible mechanisms for inducing defects in insulin signaling and secretion remain to be explored, including the role of ubiquitylation in insulin receptor internalization and trafficking. PUBLICATION HISTORY : Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis.

A ubiquitin protein ligase (E3), E3(Histone)/LASU1 (Mule/ARF-BP1/HUWE1), was recently identified that mediates ubiquitination of core histones, the Mcl-1 anti-apoptotic protein, and the p53 tumor suppressor protein. However, the expression of E3(Histone)/LASU1 remains poorly studied. Because we identified E3(Histone)/LASU1 from the testis, we explored its regulation during spermatogenesis. In the first wave of rat spermatogenesis, E3(Histone)/LASU1 mRNA and protein had peak expression at days 10 and 20, respectively, and decreased with age. Consistent with these findings, immunohistochemistry revealed that E3(Histone)/LASU1 was highly expressed in nuclei from spermatogonia to mid-pachytene spermatocytes. There was no obvious staining in spermatids, when histones are ubiquitinated and degraded. E3(Histone)/LASU1 was also expressed in other tissues. However, except in neuronal cells of the brain, expression was cytoplasmic. Thus, E3(Histone)/LASU1 may play a role in chromatin modification in early germ cells of the testis, but also has functions in other tissues.

BH3-ligand regulates access of MCL-1 to its E3 ligase.

A genome wide search for new BH3-containing Bcl-2 family members was conducted using position weight matrices (PWM) and identified a large (480kDa), novel BH3-only protein, originally called LASU1 (now also known as Ureb-1, E3(histone), ARF-BP1, and Mule). We demonstrated that LASU1 is an E3 ligase that ubiquitinated Mcl-1 in vitro and was required for its proteasome-dependent degradation in HeLa cells. Of note, the BH3 domain of LASU1 interacted with Mcl-1 but not with Bcl-2 or Bcl-Xl. A competing BH3-ligand derived from Bim interacted with Mcl-1 and prevented its interaction with LASU1 in HeLa cells, causing elevation of the steady-state levels of Mcl-1. This suggests that the unliganded form of Mcl-1 is sensitive to LASU1-mediated degradation of Mcl-1.